Regulation of eukaryotic gene expression at the transcriptional level can most productively be studied in an organism that is available in large quantities, from which RNA polymerases can be easily isolated and with characterized mutants affecting regulatory processes. The simple eukaryote, Saccharomyces cerevisiae, or baker's yeast, satisfies these criteria. Yeast contains three RNA polymerases, designated I, II and III. RNA polymerase II transcribes the genes coding for messenger RNA (mRNA). The structures, properties and specificities of yeast RNA polymerase II are analogous to those of all other eukaryotes including humans. This proposal outlines methods for isolation and characterization of the components necessary for selective transcription of yeast genes by RNA polymerase II. Templates will be prepared from clonally isolated DNA including a yeast gene which is constitutively expressed (HIS3) and an inducible gene cluster (GAL1, 7 and 10) which is regulated by other gene products (GAL4 and GAL80). Interaction of the RNA polymerase and the DNA will be examined by electron microscopic analysis of bound complexes and by comparison of in vitro transcription products to the in vivo RNAs. Soluble extracts from wild type and mutant cells under appropriate growth conditions will be combined with purified enzymes and templates; the factors required for accurate transcription will be fractionated.